A. The Rise of Tuberculosis
Over the past few years the editors of the Morbidity and Mortality Weekly Report have chronicled the unexpected rise in tuberculosis cases. It has been estimated that worldwide there are one billion people infected with M. tuberculosis, with 7.5 million active cases of tuberculosis. Even in the United States, tuberculosis continues to be a major problem especially among the homeless, Native Americans, African-Americans, immigrants, and the elderly. HIV-infected individuals represent the newest group to be affected by tuberculosis. Of the 88 million new cases of tuberculosis expected in this decade approximately 10% will be attributable to HIV infection.
The emergence of multi-dug resistant strains of M. tuberculosis has complicated matters further and even raises the possibility of a new tuberculosis epidemic. In the U.S. about 14% of M. tuberculosis isolates are resistant to at least one drug, and approximately 3% are resistant to at least two drugs. M. tuberculosis strains have even been isolated that are resistant to all seven drugs in the repertoire of drugs commonly used to combat tuberculosis. Resistant strains make treatment of tuberculosis extremely difficult: for example, infection with M. tuberculosis strains resistant to isoniazid and rifampin leads to mortality rates of approximately 90% among HIV-infected individuals. The mean time to death after diagnosis in this population is 4-16 weeks. One study reported that of nine immunocompetent health care workers and prison guards infected with drug resistant M. tuberculosis, five died. The expected mortality rate for infection with drug sensitive M. tuberculosis is 0%.
The unrelenting persistence of mycobacterial disease worldwide, the emergence of a new, highly susceptible population, and the recent appearance of drug resistant strains point to the need for new and better prophylactic and therapeutic treatments of mycobacterial diseases.
B. Tuberculosis and the Immune System
Infection with M. tuberculosis can take on many manifestations. The growth in the body of M. tuberculosis and the pathology that it induces is largely dependent on the type and vigor of the immune response. From mouse genetic studies it is known that innate properties of the macrophage play a large role in containing disease (1). Initial control of M. tuberculosis may also be influenced by reactive .gamma..delta. T cells. However, the major immune response responsible for containment of M. tuberculosis is via helper T cells (Th1) and to a lesser extent cytotoxic T cells (2). Evidence suggests that there is very little role for the humoral response. The ratio of responding Th1 to Th2 cells has been proposed to be involved in the phenomenon of suppression.
Th1 cells are thought to convey protection by responding to M. tuberculosis T cell epitopes and secreting cytokines, particularly interferon-.gamma., which stimulate macrophages to kill M. tuberculosis. While such an immune response normally clears infections by many facultative intracellular pathogens, such as Salmonella, Listeria or Francisella, it is only able to contain the growth of other pathogens such as M. tuberculosis and Toxoplasma. Hence, it is likely that M. tuberculosis has the ability to suppress a clearing immune response, and mycobacterial components such as lipoarabinomannan are thought to be potential agents of this suppression. Dormant M. tuberculosis can remain in the body for long periods of time and can emerge to cause disease when the immune system wanes due to age or other effects such as infection with HIV-1.
Historically it has been thought that one needs replicating Mycobacteria in order to effect a protective immunization. An hypothesis explaining the molecular basis for the effectiveness of replicating mycobacteria in inducing protective immunity has been proposed by Orme and co-workers (3). These scientists suggest that antigens are pinocytosed from the mycobacteria-laden phagosome and used in antigen presentation. This hypothesis also explains the basis for secreted proteins effecting a protective immune response.
Antigens that stimulate T cells from M. tuberculosis infected mice or from PPD-positive humans are found in both the whole mycobacterial cells and also in the culture supernatants (3, 4, 5-7, 34). Recently Pal and Horwitz (8) were able to induce partial protection in guinea pigs by vaccinating with M. tuberculosis supernatant fluids. Similar results were found by Andersen using a murine model of tuberculosis (9). Other studies include reference nos. 34, 12. Although these works are far from definitive they do strengthen the notion that protective epitopes can be found among secreted proteins and that a non-living vaccine can protect against tuberculosis.
For the purposes of vaccine development one needs to find epitopes that confer protection but do not contribute to pathology. An ideal vaccine would contain a cocktail of T-cell epitopes that preferentially stimulate Th1 cells and are bound by different MHC haplotypes. Although such vaccines have never been made there is at least one example of a synthetic T-cell epitope inducing protection against an intracellular pathogen (10). It is an object of this invention to provide M. tuberculosis DNA sequences that encode bacterial peptides having an immunostimulatory activity. Such immunostimulatory peptides will be useful in the treatment, diagnosis and prevention of tuberculosis.